Liver glycogenosis due to phosphorylase kinase deficiency: PHKG2 gene structure and mutations associated with cirrhosis
Liver glycogenosis due to phosphorylase kinase deficiency: PHKG2 gene structure and mutations associated with cirrhosisBarbara Burwinkel1, Susumu Shiomi2, Abdullah Al Zaben3 and Manfred W. Kilimann4,*
1Institut für Physiologische Chemie, Medizinische Fakultät, Ruhr-Universität Bochum, D-44780 Bochum, Germany, 2Third Department of Internal Medicine, Osaka City University Medical School, Osaka 545, Japan and 3Department of Pediatrics, King Fahad National Guard Hospital, Riyadh 11426, Saudi Arabia
Received September 29, 1997;Revised and Accepted October 21, 1997
Mutations in three different genes of phosphorylase kinase (Phk) subunits, PHKA2, PHKB and PHKG2, can give rise to glycogen storage disease of the liver. The autosomal-recessive, liver-specific variant of Phk deficiency is caused by mutations in the gene encoding the testis/liver isoform of the catalytic [gamma] subunit, PHKG2. To facilitate mutation detection and to improve our understanding of the molecular evolution of Phk subunit isoforms, we have determined the structure of the human PHKG2 gene. The gene extends over 9.5 kilonucleotides and is divided into 10 exons; positions of introns are highly conserved between PHKG2 and the gene of the muscle isoform of the [gamma] subunit, PHKG1. The beginning of intron 2 harbors a highly informative GGT/GT microsatellite repeat, the first polymorphic marker in the PHKG2 gene at human chromosome 16p11.2-p12.1. Employing the gene sequence, we have identified homozygous translation-terminating mutations, 277delC and Arg44ter, in the two published cases of liver Phk deficiency who developed cirrhosis in childhood. As liver Phk deficiency is generally a benign condition and progression to cirrhosis is very rare, this finding suggests that PHKG2 mutations are associated with an increased cirrhosis risk.
Glycogen storage disease of the liver due to phosphorylase kinase (Phk) deficiency can be caused by mutations in three genes coding for Phk subunits and isoforms: the liver isoform of the [alpha] subunit, [alpha]L (gene symbol, PHKA2), the [beta] subunit (PHKB) or the testis/liver isoform of the [gamma] subunit, [gamma]TL (PHKG2). These three subtypes of liver Phk deficiency differ in mode of inheritance and biochemical tissue involvement. Mutations in the PHKA2 gene cause X-linked recessive liver-specific Phk deficiency (1 -5 ), PHKB mutations cause autosomal-recessive combined Phk deficiency of liver and muscle (6 ,7 ) and PHKG2 mutations cause autosomal-recessive liver-specific Phk deficiency (8 ). As they affect an X-linked gene, PHKA2 mutations account for the large majority of liver Phk deficiency cases. Mutations in the PHKB gene have been identified in six patients; four of them are compound-heterozygotes without parental consanguinity, indicating that mutant PHKB alleles are also relatively abundant in the population. Mutations in PHKG2 seem to be the rarest variant, with only three patients described to date, all of them homozygous offspring of consanguineous parents.
The clinical appearance of liver Phk deficiency with mutations in these three genes is similar. Most patients present as infants with a protuberant abdomen because of hepatomegaly, and with growth retardation, delayed motor development and elevation of triglycerides, cholesterol and transaminases. Fasting hypoglycemia and a characteristic rounded face (`dollface') are observed in a minority of patients. Muscle weakness is often observed also in the liver-specific deficiencies with PHKA2 and PHKG2 mutations, presumably due to secondary mechanisms, whereas, conversely, muscle symptoms may be lacking even in subjects with liver and muscle Phk deficiency due to PHKB mutations. The presence or absence of clinical muscle involvement is therefore inconclusive. The prognosis is normally good. Typically, patients improve with age and become asymptomatic as adults, reaching normal height at about the age of 20 years. The phenotype of PHKB mutations seems to be particularly mild, both biochemically and clinically (6 ,7 ).
The development of liver fibrosis and even cirrhosis in Phk deficiency has been reported but seems to be very rare. Hers et al. (9 ) mention two cases of liver cirrhosis with esophageal varices and three deaths from liver adenomas and malignant tumors in a series of 205 patients with deficiency of the phosphorylase system, most of them presumably with Phk deficiency. Two cases of cirrhosis developing in childhood in association with documented liver Phk deficiency were later published (10 ,11 ). In contrast, liver cirrhosis develops almost invariably in infants with type IV glycogenosis (branching enzyme deficiency); in type III glycogenosis (debranching enzyme deficiency), liver fibrosis is characteristic but rarely progresses to cirrhosis (9 ,12 ).
In this study, we have determined the structure of the human PHKG2 gene and, based on these data, performed a molecular-genetic analysis of the two published cases of liver Phk deficiency with cirrhosis (10 ,11 ). In both, we found translation-terminating mutations in the PHKG2 gene. Together with a case of severe fibrosis among the first three PHKG2 cases reported (8 ), and given the otherwise rare occurrence of PHKG2 mutations in the patient population with liver Phk deficiency, this finding suggests that in liver Phk deficiency, PHKG2 mutations are associated with an increased risk of fibrosis and cirrhosis.
Previously, the gene of the muscle isoform of the Phk [gamma] subunit, PHKG1, was characterized from mouse and rat and shown to be organized into 10 exons extending over 15 kilonucleotides (knt) of genomic DNA (13 ,14 ). Assuming that the PHKG2 gene may be of similar organization and size as the gene of its isoform, we aimed to amplify it directly from genomic DNA by analogously dividing the published human PHKG2 cDNA sequence (15 ) into 10 putative exons and designing pairs of PCR primers corresponding to the distal ends of neighboring exons. With these primers, we could indeed amplify the complete PHKG2 gene, between the extreme ends of the cDNA, in 10 pieces overlapping at their ends within the exon sequences. The overall organization of PHKG2 is given in Figure 1 and its sequence in Figure 2 .
There is a complex microsatellite repeat at the beginning of intron 2. A series of GGT (interspersed with GCT) and GT repeats ends on CTTTC (doubly underlined in Fig. 2 ) and is partially duplicated. The sequence of this region given in Figure 2 was obtained from a homozygous individual and comprises 72 trinucleotide and 22 dinucleotide units. To investigate whether this region can serve as a polymorphic marker within the PHKG2 gene, we amplified it with primers [gamma]i20 and [gamma]i21 from 10 unrelated individuals. High allelic heterogeneity was observed, with sizes of PCR products ranging between 310 and 360 nt. Already when resolved only on a simple 1.5% agarose gel, different alleles within one sample and different band patterns of different samples could be discriminated in eight of 10 cases each. Two samples giving closely spaced doublets on an agarose gel were analyzed by radioactive labeling and electrophoresis on a denaturing gel, yielding sizes of 312/318 and 339/351 nt, respectively. The sequence in Figure 2 , obtained from one of the two samples giving a single sharp band in agarose gel electrophoresis, yields a size of 324 nt for this interval. Moreover, sequencing of another sample (the 312/318 doublet) showed that in addition to length polymorphism there is sequence heterogeneity due to GGT <-> GCT replacements (data not shown). Though we have not performed a quantitative analysis of polymorphism information content, these observations indicate a very high degree of allelic heterogeneity.
Two patients with liver Phk deficiency and cirrhosis, both daughters of consanguineous parents, were analyzed for mutations in the PHKG2 gene. Each of them was found to be homozygous for a single sequence abnormality. Patient A had a deletion of a cytosine residue in codon 93 (exon 4; Fig. 3 A), leading to a frameshift after 23% of the coding sequence and translation termination after 17 additional codons. Patient B had a C -> T transition in codon 44 (exon 3) resulting in an Arg44ter nonsense mutation (Fig. 3 B). In the earlier biochemical analysis of her family, her father and two siblings had Phk activities in the heterozygote range whereas her mother had normal Phk activity in repeated tests, so that a new maternal mutation was suspected in spite of her parents' consanguinity (11 ). However, when we analyzed her parents' DNA, we found them both heterozygotic for the nonsense mutation. This indicates that the mother's normal Phk activity had been misleading and could not rule out that she is actually a carrier of the mutation.
PCR was performed in 50 µl volumes containing 200-300 ng of genomic DNA, 300 µM of each dNTP and 150 ng of each primer. Primer sequences are available upon request. To bridge the long introns, we employed 2 U of expand long template DNA polymerase (Boehringer Mannheim) in the manufacturer's buffer 1, and for other PCRs 0.5 U of Goldstar DNA polymerase (Eurogentec) in 20 mM ammonium sulfate, 0.01% Tween-20, 1.5 mM MgCl2, 75 mM Tris-HCl (pH 9.2). Amplification was performed in 45 cycles following a step-down protocol. After denaturation for 40 s at 96°C, annealing (40 s) was carried out at a temperature 2°C below the calculated primer melting temperatures in the first five cycles, 4°C below in the next 10 cycles and 6°C below in the final 30 cycles. Elongation was performed at 68°C for 3 min in the first five cycles, for 3.5 min in the next 10 cycles and for 4 min in the final 30 cycles. Control reactions with only one primer of each pair were performed to identify unspecific side products. After electrophoresis on low-melting-point agarose gels, PCR products were sequenced directly from gel slices using the DyeDeoxy Cycle Sequencing kit from ABI essentially as described (5 ). The exons, the short introns, and ~50 nt at the ends of the long introns 2, 4 and 6 were sequenced in both directions, whereas the main parts of introns 2 and 4 were sequenced in one direction by primer walking. Moreover, most of the gene sequence was obtained from DNA of two or more unrelated individuals, except the central regions of intron 2 (nt 629-1320) and intron 4 (nt 3700-4900) which were sequenced from one person only.
The repeat region was amplified with the primers [gamma]i20 [5'-(nt 563)-TTTGAAGGCTGGAGAGTTCG-3'] and [gamma]i21 [5'-(nt 887)-CTATCCAGTCTTCTCCTGAG-3'] (Fig. 2 ). PCR without radioactive label was performed in 50 µl containing 20-50 ng of genomic DNA, 200 µM of each dNTP and 300 ng of each primer, employing Goldstar DNA polymerase (Eurogentec) and buffer as described above. Initial denaturation was at 96°C for 60 s, followed by 10 cycles of 96°C for 45 s, 58°C for 40 s, 72°C for 60 s, and finally by 35 cycles of 96°C for 45 s, 56°C for 40 s, 72°C for 60 s. Products were visualized on a 1.5% agarose gel. Amplification with radioactive label was carried out under the same conditions, except the following parameters: 10 µl volumes containing 20-50 ng of genomic DNA, 5% dimethylsulfoxide, 60 ng of each primer and 1 µCi of [[alpha]-32P]dCTP (in addition to 200 µM cold dNTPs), and an annealing temperature of 56°C in all 45 cycles. Products were resolved on a 6% polyacrylamide sequencing gel with an M13 sequencing ladder standard and visualized by overnight autoradiography of the dried gel at room temperature.
Detailed descriptions of both patients have been published. Patient A (EO.O; ref. 10 ) is the daughter of healthy Japanese parents who are first cousins. She was found to have adenomatous hyperplasia and cirrhosis of the liver at the age of 15 years. She had short stature and abdominal swelling as a young child but eventually grew to almost normal height. Histological examination of a liver biopsy showed glycogen storage. Phk activity in erythrocytes was 10% the value of a healthy subject. The change in blood glucose with glucagon stimulation was blunted after a meal, and absent when the patient was fasting. Today, with the patient aged 26 years, the liver tumor has not changed in size, but the cirrhosis has progressed, with the development of esophageal varices and ascites.
Patient B (BA.R; ref. 11 ) is the daughter of healthy consanguineous Saudi-Arabian parents (first cousins); one brother and one sister are healthy. She came to medical attention at 2 and 4 months of age with early-morning seizures and abdominal extension. At 11 months, she was admitted in a semi-comatose condition after a seizure, and presented with massive hepatomegaly, growth retardation (weight and length at the 5th percentile), low blood glucose (1.8 mM) and high serum lactate, transaminases and triglycerides. Liver glycogen was markedly elevated (20%; normal, 2-6%). Phk activity was undetectable in liver and erythrocytes but normal in muscle. The phosphorylase a/a + b ratio in liver was low (0.1; normal, 0.4-0.5). During follow-up until age 5.5 years, recurrent seizures, hypoglycemia, transaminases fluctuating between five and 25 times the normal values, lactic acidosis and a flat glucagon response were observed. Sequential liver biopsies showed progression to cirrhosis. Now, at the age of 8 years, she has not recently had hypoglycemic symptoms or convulsions, but continues to be shorter than normal (below the 5th percentile).
PCR primers (A-D) and additional sequencing primers (S) for mutation analysis of the PHKG2 gene
PCR/Seq.
Primer
Position/direction
Sequence
A
[gamma]TL F1
-89a
>
5'-TGAGCGACTGCAGGCAAAC
A
[gamma]i 21
Ex2 +387
<
5'-CTATCCAGTCTTCTCCTGAG
S
[gamma]TL AR
Ex2 +66
<
5'-CAAACGGAAGCGGGAATGC
B
[gamma]TL BF
Ex3 -56
>
5'-TGTGCGGAAATGTGAGCAC
B
[gamma]TL BR
Ex4 +67
<
5'-CACAAATGCTGGGCCATAC
C
[gamma]TL CF
Ex5 -67
>
5'-GCACTGGTCTGTTTTCTCAG
C
[gamma]TL CR
Ex6 +66
<
5'-AGCCAACCAATCTCATCAGG
D
[gamma]TL FD
Ex7 -71
>
5'-AGCAGAGATCCAGTGCTAAG
D
[gamma]TL R3
1293a
<
5'-CAGAGGAGCTGGAATGATCA
S
[gamma]TL H2
808a
>
5'-AGGCTGCTGCAGGTGGATC
S
[gamma]TL R2
875a
<
5'-TCAAAGAAGGGGTGCTGTAG
aThese primers lie within exons. Nucleotide positions therefore refer to the cDNA sequence.
Genomic DNA was isolated from whole blood, and four regions of the PHKG2 gene that encompass the complete coding sequence (exon 2, exons 3 and 4, exons 5 and 6, and exons 7-10) were amplified and analyzed by direct sequencing essentially as described previously (5 ). Primers for PCR and sequencing are given in Table 2 .
We thank Dr Olaf Riess and Ms A.M. Menezes Vieira-Saecker (Bochum) for help with the microsatellite repeat characterization and for DNA from CEPH individuals. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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B. BURWINKEL, M S. TANNER, and M. W KILIMANN Phosphorylase kinase deficient liver glycogenosis: progression to cirrhosis in infancy associated with PHKG2 mutations (H144Y and L225R)
J. Med. Genet.,
May 1, 2000;
37(5):
376 - 377.
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